The standard treatment for metastatic renal cell carcinoma (RCC) includes cytokine therapy with interferon-alpha (IFN-α) or interleukin-2 (IL2), which produce 15-20% response rates. These modest response rates highlight the need for more effective treatments; however, they also indicate that RCC is immunoresponsive. An effective tumor vaccine targets antigens that are highly expressed on tumor cells. Recombinant heat shock proteins (HSP) can be used to stimulate the immune system to target tumor-specific antigens, leading to tumor killing. HSP are one of the most abundant proteins found inside a cell. They have the ability to bind and protect intracellular proteins in the presence of cellular stress, such as heat and glucose depravation.
Recombinant HSP, such as hsp100 and grp170, can be complexed to a tumor-specific antigen. Depending on the tumor antigen, the HSP-target antigen complex forms at room temperature or when heated. The complex is then administered as a vaccine that targets the tumor.
Heat shock proteins (HSP) are some of the most abundant intracellular proteins. They normally function as molecular chaperones, assisting with protein folding and formation of multi-subunit complexes. They are induced by cellular stress and protect intracellular proteins by binding and preventing denaturation. HSP are broadly categorized as hsps (designated here using small characters) or glucose regulated proteins (grps) based on their subcellular localization and the stressors that induce their expression ((Shen, J. et al., Coinduction of glucose-regulated proteins and doxorubicin resistance in Chinese hamster cells. Proc Natl Acad Sci USA 84, 3278-82 (1987)) ((Srivastava, P., Roles of heat-shock proteins in innate and adaptive immunity. Nat Rev Immunol 2, 185-94 (2002)). For example, hsps (families of hsps: small hsps, hsp40, calreticulin, hsp60, hsp70, hsp90, hsp110) are induced by heat and oxidizing agents, and localize to the nucleus, cytoplasm and mitochondria. Grps (family of grps: grp78, grp94, grp170) are induced by hypoxia and glucose deprivation, and localize to the endoplasmic reticulum.
Experiments performed in the early 1900s demonstrated that tumor cells and lysates can protect mice against subsequent tumor challenges. Follow up experiments using tumor fractions identified HSP as the “active ingredient” providing immune protection (Udono, H. & Srivastava, P. K., Comparison of tumor-specific immunogenicities of stress-induced proteins gp96, hsp90, and hsp70. J Immunol 152, 5398-403 (1994)). The HSP may be promiscuously bound to a number of tumor antigens, which may produce a tumor-specific immune response; although, it is not yet possible to specifically predict with any certainty that that such a response will in fact occur with a particular HSP bound to a particular antigen without actual tests (Castelli, C. et al. Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells. Cancer Res 61, 222-7 (2001)).
It has been postulated that HSP found outside a cell are recognized as a danger signal, indicating to the immune system the presence of damaged or diseased tissue. Receptors for HSP have been identified on dendritic cells (DC), which are professional antigen presenting cells (APC). Using solubilized APC membrane applied to a gp96 affinity column, CD91 was identified as a receptor for HSP; CD91 binds hsp90, hsp70 and calreticulin. Various scavenger receptors including CD14, TLR-2 and TLR4 have been shown to bind and internalize hsp70 and hsp60. The binding of HSP and DC leads to NF-κB signaling, which has previously been shown to regulate cytokines and DC maturation.
In certain cases, microgram quantities of HSP bound to peptides may serve as a powerful immune adjuvant, activating both an antigen-specific and an innate immune response. While the majority of exogenous antigens produce a MHC class II response, proteins and peptides bound to HSP may elicit a MHC class I mediated CD8+ T cell response as well as a MHC class II response (Udono, H., Levey, D. L. & Srivastava, P. K., Cellular requirements for tumor-specific immunity elicited by heat shock proteins: tumor rejection antigen gp96 primes CD8+ T cells in vivo. Proc Natl Acad Sci USA 91, 3077-81 (1994)) (Matsutake, T. & Srivastava, P. K., The immunoprotective MHC II epitope of a chemically induced tumor harbors a unique mutation in a ribosomal protein. Proc Natl Acad Sci USA 98, 3992-7 (2001)). The mechanism of cross presentation is the subject of active research; however, it known that cross presentation of peptides bound to HSP requires functional proteosomes and transporter associated with antigen processing (TAP). HSP uncomplexed to peptide might stimulate an innate immune response by stimulating the secretion of various cytokines including, TNFα, IL-1α, IL-6, IL-12, and GM-CSF (Basu, S., Binder, R. J., Suto, R., Anderson, K. M. & Srivastava, P. K., Necrotic but not apoptotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway. Int Immunol 12, 1539-46 (2000).) (Kol, A., Lichtman, A. H., Finberg, R. W., Libby, P. & Kurt-Jones, E. A., Cutting edge: heat shock protein (HSP) 60 activates the innate immune response: CD14 is an essential receptor for HSP60 activation of mononuclear cells. J Immunol 164, 13-7 (2000)). Both the antigen-specific and the innate immune responses contribute to the final anti-tumor effect.
HSP vaccine strategies have been reported by others. HSPs are complexed to a wide spectrum of intracellular tumor proteins. It is therefore possible to isolate these HSPs and administer them as a tumor-specific, autologous vaccine. In principle, this approach is similar to using tumor lysate as a vaccine; however, the extraction of tumor HSPs produces a more concentrated vaccine enriched for the “active ingredient”. Using this approach, a phase III clinical trial for metastatic melanoma and a phase III adjuvant therapy trial for kidney cancer have completed enrollment. In two different phase II trials for metastatic kidney cancer, approximately 35% of patients had a clinical response. No significant toxicities were observed, and no autoimmune effects were noted (Amato, R. et al. in ASCO A1782 (2000)) (Assikis, V. J. et al. in ASCO A1552 (2003)).
There unfortunately are limitations to using tumor derived HSPs. Surgically obtained tumor tissue is not available for all patients. Even when tumor tissue is available, a vaccine can not be prepared in approximately 10% of cases. Finally, only a small fraction of relevant tumor peptides in the vaccine produce an antitumor effect. Therefore, in an effort to produce a highly concentrated vaccine against a known tumor antigen, genetically engineered proteins consisting of HSP fused to the C or N terminus of a tumor protein were synthesized (Udono, H., Yamano, T., Kawabata, Y., Ueda, M. & Yui, K., Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70. Int Immunol 13, 1233-42 (2001)) (Suzue, K., Zhou, X., Eisen, H. N. & Young, R. A., Heat shock fusion proteins as vehicles for antigen delivery into the major histocompatibility complex class I presentation pathway. Proc Natl Acad Sci U S A 94, 13146-51 (1997)) (Anthony, L. S. et al., Priming of CD8+ CTL effector cells in mice by immunization with a stress protein-influenza virus nucleoprotein fusion molecule. Vaccine 17, 373-83 (1999)). In most cases the HSP is of microbial origin and can itself produce an immune response. Although an unlimited supply of vaccine can be produced, this approach does not produce an immune response in all cases. A HPV 16-E7 (cervical cancer antigen)-hsp110 fusion vaccine was, for example, created that did not produce a CD8+ CTL response in vivo (unpublished data). A possible explanation for this negative result is that a fusion protein is an unnatural construct and interactions with APC depend on proper positioning and steric changes associated with noncovalent complexing of HSP and tumor antigen.